PROJECT SUMMARY Penile anaerobes are significantly reduced after male circumcision (MC), but it is unclear if these anaerobes cause genital inflammation and increase HIV susceptibility. Without this knowledge, we are unlikely to understand the role of the penile microbiome in mucosal inflammation and HIV susceptibility. Our long-term goal is to develop novel preventative strategies to augment MC. This project's objective, which is the logical next step toward our goal, is to determine the impact of penile anaerobes on HIV risk. Our central hypothesis is that penile anaerobes increase HIV susceptibility in men, irrespective of MC status, and that penile anaerobes can both stimulate local synthesis of innate immune mediators and increase recruitment and activation of HIV- susceptible cells in the penile epithelium. Our rationale for this work is that, once we know how penile anaerobes influence genital inflammation and HIV risk, we could develop novel strategies, such as narrow- spectrum antimicrobials (e.g., bacteriocins, bacteriophages) to reduce penile anaerobe load or genital inflammation. We will achieve our objectives by accomplishing the following specific aims: Aim 1: Determine the association between penile anaerobes and HIV acquisition in men using samples and epidemiologic data from the Rakai MC trial. We will directly assess whether penile anaerobes are associated with increased HIV risk, using a nested case-control study design and existing samples from seroconverters (prior to seroconversion) (n = 93) and seronegative controls from the Rakai MC Randomized- controlled trial (RCT), matched at 1:2. Aim 2: Determine the correlation between penile anaerobes and HIV target cell recruitment and activation. We will enroll 194 HIV-negative men, ages 15-49, who request MC. We will collect urethral and coronal sulcus swabs prior to MC and foreskin tissue at time of MC to analyze the penile microbiome and foreskin HIV target cell activation and density. Aim 3: Define the temporal association between penile anaerobes and local immune responses. We will sample 104 men, ages 15-49, over an 8-month period, including 52 men from Aim 2 and an additional 52 HIV-negative men who decline MC. We will begin sampling circumcised men after complete wound healing at 6 weeks post-MC. Using these longitudinal coronal sulcus swabs, we will determine penile microbiome stability and the temporal association between anaerobe and inflammatory cytokine production. The approach is innovative, in this team's opinion; because it will identify penile anaerobe strains associated with HIV seroconversion and the most potent inducers of mucosal inflammation, irrespective of MC status. The project is significant because it will elucidate role of penile microbiome in HIV acquisition and has the potential to lead to new non-surgical options for men who decline MC and novel strategies to augment MC.